Keywords

5.1 Introduction

Bluetongue disease (BT) is a non-contagious vector-borne disease that mainly affects ruminants but also camelids. It is caused by the BT virus (BTV), which belongs to the genus Orbivirus within the family Reoviridae and of which 24 serotypes are known; further serotypes awaiting confirmation were described in Switzerland (Toggenburg virus) (Hofmann et al. 2008) and Kuwait (Maan et al. 2011a, b). BTV is transmitted between hosts almost exclusively through the bites of female Culicoides biting midges. BT is a notifiable disease under the German Animal Disease Act, reportable in the European Union via the Animal Disease Notification System (ADNS) and at the global level notifiable to the World Organisation for Animal Health (OIE).

BT had never been reported in Germany before it occurred in the region of Aachen in North Rhine-Westphalia in August 2006, almost simultaneously with outbreaks in Belgium and the Netherlands (EU Rapid Press release Nr. IP/06/1112, ProMedMail, 20060828.2448, Mehlhorn et al. 2007; Toussaint et al. 2007).

5.2 Pathogenesis

BTV is spread by infected haematophagous insects, mainly biting midges (Culicoides spp.) that excrete the virus in their saliva. In addition to the transmission itself, it has been suggested that immunomodulatory proteins in the midge saliva aid in the initial infection of the host (Darpel et al. 2009). During a blood meal, BTV is inoculated into the skin, which may be both an important site for replication and a source of virus for blood-feeding vectors (Darpel et al. 2009). After inoculation, migrating dendritic cells transport the virus to the draining lymph node (Barratt-Boyes et al. 1995; Hemati et al. 2009), from where it disseminates to other lymphoid tissues (Pini 1976; Barratt-Boyes and Maclachlan 1994). Replication occurs principally in mononuclear phagocytic cells, proliferating lymphocytes and endothelial cells (Mahrt and Osburn 1986; Maclachlan et al. 1990; Barratt-Boyes et al. 1992; DeMaula et al. 2001; Drew et al. 2010b). Accordingly, bluetongue pathogenesis is characterized by virus-mediated immune suppression, endothelial injury and dysfunction (DeMaula et al. 2002a; Maclachlan 2004; Maclachlan et al. 2009; Umeshappa et al. 2010).

Virus replication in endothelial cells causes direct cell injury and necrosis. Vascular blockage leads to haemorrhage and tissue infarction (Mahrt and Osburn 1986) that can manifest as myonecrosis and mucosal ulceration (Drew et al. 2010b). The activation of pulmonary endothelial cells and macrophages (DeMaula et al. 2002a, b; Drew et al. 2010b), on the other hand, and the subsequent release of host-derived inflammatory and vasoactive mediators (Hemati et al. 2009) increase vascular permeability, potentially leading to the widespread oedema often seen in fatal BT, African horse sickness and other virus-induced haemorrhagic fevers (Maclachlan et al. 2009; Drew et al. 2010a; Maclachlan 2011). A confirmation of this hypothesis, however, will require a deeper understanding of the highly complex interplay of cytokines in infected ruminants beyond the isolated findings of groundbreaking in vitro studies.

5.3 Clinical Signs

Before the first outbreaks of BT in Central Europe in 2006, clinical signs of the disease were mainly described for sheep (Erasmus 1990). However, it is known since many years that BTV can infect several domestic and wild ruminant species (Tabachnick 1996; Darpel et al. 2007). The severity of the disease in sheep may depend to a marked extent on environmental conditions, most notably exposure to sunlight, a frequently ignored fact (Erasmus 1990). Other authors doubt whether there is an ill-defined interaction with the environment (Mellor and Wittmann 2002). The clinical picture also varies depending on the strain of the virus as well as the breed and age of the infected animals with older age groups being more susceptible (Tabachnick 1996; Mellor and Wittmann 2002). In general, the clinical picture of BT can be extremely variable (EFSA 2007).

5.3.1 Sheep

Irrespective of the region of the world from where BTV serotypes originate, clinical disease of sheep follows a similar pattern (Parsonson 1992). The severity of clinical signs depends on both the breed of sheep and the strain of virus (Darpel et al. 2007). All breeds of sheep are susceptible to BTV infection, although the clinical outcome may vary remarkably. Especially indigenous African breeds have been reported as resistant (Erasmus 1990). However, not only African but also other indigenous breeds seem to be less susceptible than introduced European breeds and Merino sheep (Parsonson 1992) or may only be affected subclinically (Darpel et al. 2007). Febrile reactions with fever exceeding 41°C are common (Erasmus 1990). This could also be reproduced with a BTV-8 strain in experimental infections (Darpel et al. 2007). Within 1–2 days after the onset of the disease, the skin of the muzzle and lips as well as the oral mucosa became hyperaemic and oedematous (Erasmus 1990). This was also frequently observed during the BTV-8 epidemic in Central Europe between 2006 and 2009. Figure 5.1 shows a ewe from a backyard farm in North Rhine-Westphalia in September 2006. During the BTV-8 outbreak in Germany 2006–2009, clinical disease was often very severe in sheep.

Fig. 5.1
figure 1

Ewe with hyperaemic muzzle and shallow erosions and crusts on the nostril

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Nasal discharge, which became later sometimes mucopurulent, and resulting dyspnoea could often be observed.

Foot lesions developing with the subsidence of fever represented a frequently detected disease manifestation. The coronary band is hyperaemic often combined with petechial haemorrhages.

An investigation in sheep farms in 2007 revealed that stillborn lambs showed clinical signs such as crusts and lesions of the oral mucosa (Fig. 5.2a). One of the farmers reported that the newborn lambs were physically and motorically retarded for several weeks. Feet lesions were apparently so painful that some of the sheep were reluctant to walk for weeks even after the acute symptoms had healed. Instead, they crawled on their carpal joints (Fig. 5.2b). When the animals were supported or when they got shooed, they walked on their feet for a little while; however, they immediately fell back to their previous behaviour when they were left alone.

Fig. 5.2
figure 2

Clinical signs of BT in sheep; (a) lips and tongue of a stillborn lamb with crusts and lesions in the oral mucosa; (b) sheep walking on carpal joints

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5.3.2 Goats

Reports on clinical signs with BTV-8 virus strains in goats are rare. During experimental studies with BTV-8 in Dutch dairy goats, fever, signs of general illness, apathy, dysphagia, diarrhoea and lameness were observed (Backx et al. 2007). The discrepancy between the observations in the field and in laboratory experiments might be explained by different routes of infection, e.g. intravenous injection vs. the natural route of infection via biting midges or by vector preferences or the types of husbandry systems (Backx et al. 2007). During the BTV-8 epidemic in Germany in 2006–2009, a total of 26,954 BTV-8-infected premises were recorded (TSN database; 15.11.2011, 1135 hours), among which there were 132 holdings where goats were reported as clinically affected (TSN database; 15.11.2011, 1135 hours). This shows that goats were affected by this epidemic in Germany, but only to a limited extent.

5.3.3 Cattle

It is thought that cattle have now largely replaced antelopes as a maintenance host of the virus in Africa (Gerdes 2004). However, before the BTV-8 epidemic in northern Europe, natural and experimental BTV infection of cattle was considered asymptomatic in the vast majority of cases (Maclachlan et al. 1992; Maclachlan 2011). A transient febrile response, lacrimation and salivation were occasionally observed in infected animals (Erasmus 1990).

At the beginning of the BTV-8 epidemic in Central Europe, clinical symptoms such as fever (40–41°C) for 2–14 days, severe nasal discharge, lacrimation, facial oedema and nasal excoriations were described in adult cattle (Mehlhorn et al. 2007). However, using a field virus strain that originated from a BT outbreak in the Netherlands in an experimental infection, no pyrexia was recorded in any of the cattle (four 6-month-old male Holstein-Friesian calves) at any stage of the experiment (Darpel et al. 2007). One of the first German BTV-8 outbreaks was detected in a beef suckler herd (57 cattle, Limousin breed) in North Rhine-Westphalia. One cow showed a bilateral conjunctivitis, oedema of the eyelids combined with petechial haemorrhages on the swollen mucous membranes of the eyelids. Additionally, strong lacrimation was obvious as a predominant clinical sign (Fig. 5.3). The local tissue damage was complicated by bacterial infection.

Fig. 5.3
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Limousin cow with swollen eyelids, lacrimation and petechial haemorrhages

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Ulcers and erosions may occur in the oral mucosa (Fig. 5.4). The skin of the muzzle was inflamed at the beginning of the disease; later, cracks and peels could be observed (Fig. 5.5).

Fig. 5.4
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Erosion on the oral mucosa in healing after BTV-8 infection

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Fig. 5.5
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Erosions, hemorrhagic lesions accompanied by bacterial superinfections and inflammatory foci of on the muzzle of a BTV-infected cattle

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Large-scale erosions or haemorrhagic lesions in the skin of the teats were quite often reported in dairy cattle (Fig. 5.6). These lesions sometimes resulted in detachments of bigger parts of the skin of the affected teats. This caused pain during milking or nursing. Later on, these lesions were also affected by bacterial infection or characterized by the formation of crusts.

Fig. 5.6
figure 6

Large-scale hemorrhagic lesions on the teats of BTV-8 infected cattle

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Lesions involving coronitis and inflammation of the whole foot region were also frequently reported. The coronary bands were hyperaemic, swollen and inflamed. Therefore, stiffness or lameness is common in BTV-infected cattle (Erasmus 1990).

5.4 Diagnosis

5.4.1 BTV Isolation

For the direct detection of bluetongue virus, antigen or genome, whole blood is preferred over plasma because BTV is closely associated with red blood cells (Brewer and Maclachlan 1992; Nunamaker et al. 1992). Recommended specimens for the isolation of bluetongue virus are blood of live animals or spleen samples collected at necropsy. Heparinized or EDTA-treated blood that has been washed several times with phosphate-buffered saline to remove BTV antibodies is best suited for virus isolation. The release of virus particles from the erythrocyte membrane by sonication after washing can increase the success of the isolation. BTV-positive spleen samples are homogenized in cell culture medium and cleared by centrifugation. The purified supernatants can then be used for the inoculation of cultured cells or embryonated chicken eggs. For the latter, most laboratories use the intravenous inoculation method published by Goldsmit and Barzilai (1985). BTV-infected chicken embryos usually die within 2 and 7 days, and appear cherry red as a result of massive haemorrhages.

Besides mammalian cell lines (e.g. baby hamster kidney cells [BHK-21], African green monkey kidney cells [Vero] or bovine aorta endothelial cells), several insect-derived cell lines (e.g. Aedes albopictus clone C6/36 or Culicoides variipennis larval [KC] cells) can be used for isolation and propagation of BTV (Clavijo et al. 2000). The passage of virus in cell culture typically results in an adaptation of the virus to the in vitro conditions (Gould et al. 1989).

A highly sensitive and reliable method for the isolation of BTV is the inoculation of susceptible animals, mainly sheep or cattle. Although animal experiments are very expensive and ethical considerations have to be taken into account, an important advantage can justify this approach. Based on the large volume of inocula (up to 500 ml sample material are tolerated during intravenous injection), the sensitivity is very high. For samples with borderline infectivity (blood from animals in a late stage of infection or semen samples with a low viral load), the inoculation of susceptible animals can be the only way to propagate the virus (Hourrigan and Klingsporn 1975; Eschbaumer et al. 2010a, b).

Laboratory animals such as mice are not regularly used for BTV diagnosis and research. Nevertheless, the intracerebral inoculation of BTV isolates in 2–3 days old suckling mice causes clinical signs and death within 2–5 days after inoculation. Recently, a novel mouse model for BTV using type I interferon-receptor-deficient (IFNAR−/−) mice was developed (Calvo-Pinilla et al. 2009b; Eschbaumer et al. 2010b). Owing to the high susceptibility of these mice to fatal infection with BTV, they can also be used for virus isolation from samples harbouring a small viral load.

5.4.2 Molecular Diagnosis

Historically, confirmation and classification of BTV isolates have been based on immunological assays such as the indirect immune fluorescence test (Ruckerbauer et al. 1967; Jochim and Jones 1983), the complement fixation test (Shone et al. 1956), the haemagglutination assay (van der Walt 1980; Hübschle 1980), electron microscopy (Gould et al. 1989; Nunamaker et al. 1992), virus neutralization assays (Howell 1960) and competitive antigen-capture ELISAs (Mecham 1993; Mecham and Wilson 2004). With the development of nucleic acid detection methods in the late 1980s, cloned cDNA segments of several BTV serotypes were used to define the genetic relationships between and within the BTV serotypes (Unger et al. 1988; Ritter and Roy 1988). The introduction of the polymerase chain reaction (PCR) in the beginning of the 1990s revolutionized the molecular diagnosis of BTV (Wade-Evans et al. 1990; McColl and Gould 1991). In the following years, several improvements of BTV genome detection by PCR were published. Besides one-step RT-PCR assays, nested PCR systems and multiplex PCR systems for the identification of different BTV serotypes circulating in one region were developed (Wilson and Chase 1993; Katz et al. 1994; Aradaib et al. 1998; Zientara et al. 2004). Primers of different segments were used for the amplification and characterization of BTV strains (see Hoffmann et al. 2009a, for a review). For group-specific assays, conserved regions of the segments 5, 6, 7 and 10 were identified (Aradaib et al. 1998; Pierce et al. 1998; Bandyopadhyay et al. 1998; Anthony et al. 2007). Specific primers for the VP2 gene were developed and used in singleplex or multiplex assays to determine the BTV serotype (Wilson and Chase 1993; Eschbaumer et al. 2011b).

A new era for the molecular diagnosis of BTV began with the development of the real-time RT-PCR technology in the 1990s (Higuchi et al. 1993; Wittwer et al. 1997). Prior to the BTV-8 outbreak in Europe in the summer of 2006, only very few real-time quantitative RT-PCR (RT-qPCR) assays for the detection of BTV had been published. The first used primers were designed for the detection of the NS1 gene (Seg-5) (Wilson et al. 2004). However, this assay detected only 11 out of the 19 serotypes tested. The same year, another RT-qPCR was published using Förster resonance energy transfer (FRET) probe technology targeting genome segment 2 (VP2) (Orru et al. 2004). In 2006, an RT-qPCR assay was developed using a conserved region in RNA segment 5 of BTV-2 and BTV-4 (Jimenez-Clavero et al. 2006). This assay detected all of the recent Mediterranean isolates that were tested, BTV vaccine strains for serotypes 2 and 4 as well as 15 out of the 24 BTV reference strains. In the European outbreak of BTV-8, however, this assay showed a reduced sensitivity for the field strain of BTV-8 compared to other assays (Batten et al. 2008a). In the same year, an RT-qPCR was developed using a molecular beacon (MB) fluorescent probe designed within the NS3 conserved region of segment 10 (Orru et al. 2006).

Since the start of the northern European outbreak in August 2006, many RT-qPCR assays have been developed. Most of these were assays for the detection of all 24 serotypes identified at this time (Toussaint et al. 2007; Shaw et al. 2007), using conserved regions of the VP1 (seg-1) and NS1 (seg-5) genes. Nevertheless, the reduced sensitivity for the novel BTV serotypes 25 and 26 (Hofmann et al. 2008; Maan et al. 2011a, b) confirms the necessity for regular verification of the functionality of such pan-BTV assays. The parallel use of independent assays with equivalent diagnostic sensitivity and specificity can overcome the limitations of one assay. Another option is the application of pan-orbivirus assays, which use primer binding sites in the polymerase gene (seg-1) that are conserved among all currently known orbiviruses (Palacios et al. 2011). In addition to the group-specific (pan-BTV) real-time RT-PCR assays, serotype-specific BTV assays were developed and validated by diagnostic laboratories (Mertens et al. 2007; Hoffmann et al. 2009a, b). Furthermore, the advantages of the real-time PCR technology can be used for high-throughput analyses (Vandemeulebroucke et al. 2010). The use of robotics for automated extraction of BTV RNA combined with the co-amplification of the BTV target and an internal control RNA ensures a high diagnostic reliability during the investigation of large sample batches (Toussaint et al. 2007; Vandenbussche et al. 2010). These can be blood and tissue specimens in outbreak situations or insect samples from entomological monitoring programmes. High-throughput RNA extraction and real-time RT-PCR are particularly indispensable for the BTV analysis of extensive numbers of midges (Hoffmann et al. 2009c; Vanbinst et al. 2009).

5.4.3 Serology

Historically, BTV antibody detection in serum relied on complement fixation and agar gel immunodiffusion. Both, however, proved inferior to enzyme-linked immunosorbent assays (ELISAs) and were eventually replaced (Afshar 1994; Hamblin 2004). Highly sensitive ELISAs can pick up the humoral immune response to BTV as early as 1 week after infection (Batten et al. 2008a; Oura et al. 2009). Several systems are commercially available, mostly detecting antibodies against VP7, a BTV structural protein that is largely conserved across all serotypes. Three competitive ELISA kits (by ID VET, IDEXX and VMRD) are currently licensed for use in Germany. A latex agglutination test (Yang et al. 2010a) and immunochromatographic strips (Yang et al. 2010b) have recently been proposed, promising faster sample turnover and the possibility of pen-side testing.

Apart from the competitive ELISAs, an indirect assay is available for individual and bulk milk samples (Kramps et al. 2008; Chaignat et al. 2010; Mars et al. 2010). Double-antigen sandwich ELISAs, which use peroxidase-labelled antigen to detect captured antibody (Laman et al. 1991), display superior sensitivity early in infection and more reliably detect vaccine-induced antibody (Eschbaumer et al. 2009; Oura et al. 2009). Two kits (by ID VET and Prionics) are commercially available in Germany.Footnote 1 However, their bias for multimeric antibody molecules such as immunoglobulin (Ig) M may have a negative impact on sensitivity during the transition from IgM to IgG in the development of the immune response (Eschbaumer et al. 2011a).

Beyond the performance of a group-specific assay, serotyping usually requires labour-intensive neutralization tests against a panel of reference viruses (Hamblin 2004). A serotype-specific antibody ELISA for BTV-8 has recently been implemented, but data on its performance are not yet available.

Regardless of the test format, the widespread use of inactivated whole virus vaccines in Europe (Zientara et al. 2010) interferes with serological surveillance. The commercially available VP7-based tests are unable to differentiate between infected and vaccinated animals (Mertens et al. 2009). Theoretically, inactivated vaccines should only elicit antibody responses to structural proteins. The discrimination potential of ELISAs based on non-structural proteins NS1 or NS3 has been demonstrated (Anderson et al. 1993; Barros et al. 2009) but is highly dependent on the purity of the vaccine. The carryover of non-structural proteins from the culture system used to produce the vaccine may result in antibodies to those proteins in vaccinated animals (Alpar et al. 2009), particularly after repeated vaccinations. Recent attempts at establishing a commercial NS1 ELISA were not successful. Vaccination with inactivated vaccines from different companies led to an increased number of unspecific results in the test, which, consequently, was not released by the manufacturer.

5.5 Epidemiology

5.5.1 Disease Transmission

When BTV serotype 8 (BTV-8) first appeared in Central Europe, no data on the putative vectors were available. Transmission of BTV had generally been supposed to be associated with Culicoides imicola, the most efficient and widespread vector in the Old World (Meiswinkel et al. 2007). Since this species is restricted to Africa and southern Europe, the occurrence of BT in more northern countries and its effective transmission by indigenous vectors was surprising, despite evidence from Italy suggesting that also C. pulicaris and midges of the C. obsoletus group can harbour BTV (Caracappa et al. 2003; Savini et al. 2004).

Meanwhile, entomological monitoring has shown that C. imicola is still not present in Germany and that members of the C. obsoletus and C. pulicaris complexes are relevant vectors for BT in Germany and Central Europe (Hoffmann et al. 2009c; Mehlhorn et al. 2009a). They are small midges of approximately 1–2 mm length which tend to be active in the evening, night and dawn. The length of time between ingestion of virus by a midge and its appearance in the saliva, the so-called extrinsic incubation period, is influenced by both temperature and salivary proteases of the vectors. At temperatures of 10–30°C, the extrinsic incubation period becomes progressively shorter with increasing temperatures since:

  • Midges feed more frequently.

  • Both the reproductive cycle of midges and virus replication in midges are faster.

  • A greater proportion of the midge population becomes vector competent.

  • Possibly more midge species become vector competent (Hateley 2009).

In Germany, transmission of BT is apparently interrupted or at least significantly reduced during the cold season (late autumn, winter and early spring) as a consequence of the low temperatures, which reduce the activity of Culicoides biting midges and BTV replication in the midgut of the biting midges. However, BTV might persist during the winter either in the vector population or in the host population (Wilson et al. 2008). Another possible way of overwintering is persistence in an as yet unknown wild ruminant population, e.g. red deer. In Belgium, relatively high levels of seroprevalence have been observed in red deer during 2007 and 2008. By contrast, the seroprevalence in roe deer was low, which was explained by the fact that red deer live in large groups, move more and therefore might be more exposed to insects (Linden et al. 2010). The results of the German wildlife monitoring on BT provide no evidence for the conclusion that a reservoir for BTV might have formed in wild ruminants in Central Europe. However, the relative importance of the remaining potential overwintering mechanisms remains unclear, too (Napp et al. 2011). As active midges were also found in the cold season, although in reduced numbers and mostly close to or within stables, a true midge-free period does not exist in Germany (Mehlhorn et al. 2009b).

Several investigation programmes were set up to share European BT outbreak data, e.g. in the BT51 group and as part of EPIZONE, an EU-funded Network of Excellence. Special attention has been paid to both mechanistic and stochastic predictive modelling which included a wide range of predictor variables to assess the spread of BTV (Faes et al. 2011; de Koeijer et al. 2011; Ducheyne et al. 2011; Willgert et al. 2011).

5.5.2 Introduction of Bluetongue Disease into Germany

In 2006, the first infections with bluetongue virus of serotype 8 (BTV-8), which was initially restricted to the sub-Saharan region, Asia and South America, invaded Central Europe. On August 18th, BTV-8 was confirmed in the Netherlands (EU Rapid Press release Nr. IP/06/1112, ProMedMail, 20060828.2448) and Belgium (Toussaint et al. 2007). On 21 August 2006, the first outbreak was reported in the neighbouring district of Aachen on the German side of the border (OIE, immediate notification, Conraths et al. 2007). The virus hit an area with a high density of BT-naive animals, presence of suitable vectors (Culicoides spp.), climatic conditions favourable for virogenesis in local biting midges and for transmission. So far, the epidemiological situation in Germany has been dominated by BTV-8 (Conraths et al. 2009; Gethmann et al. 2010), but a few cases of infections with BTV-6 and a single case of BTV-1 in an imported animal were reported (Eschbaumer et al. 2010a).

In addition to entomological patterns, much research has been devoted to the determination of the most likely time and place of introduction of BTV-8 (Saegerman et al. 2010). The following hypotheses to explain the introduction of BTV-8 to Western Europe were taken into consideration:

  • Legal or illegal import of viraemic susceptible animal species.

  • Legal or illegal import of infected non-susceptible animal species, especially against the background that the World Equestrian Games took place in Aachen at the time when the first BT cases were noticed.

  • Introduction via infected vectors, especially because midges are so light that they can drift by wind over hundreds of kilometres (Ducheyne et al. 2007). Alba et al. (2004) confirmed the possibility of introduction of infected midges to the Balearic Islands from Sardinia during the BT outbreak in the year 2000. It has also been proposed that BTV-infected biting midges might have been moved from northern Africa to Spain, Portugal or Italy by wind across the Strait of Gibraltar and the Mediterranean Sea in 2006 (Gloster et al. 2007; Hendrickx 2008). Another alternative is that infected vectors might have been directly introduced to Europe, e.g. by airplane with flowers imported from regions where BTV is enzootic. This hypothesis was also considered when BTV-6 occurred in the district of Grafschaft Bentheim (Lower Saxony) on the border to the Netherlands, although the respective virus closely resembled an isolate used in an attenuated live vaccine (Eschbaumer et al. 2010a).

Despite intensive epidemiological investigations, the source of the introduction has never been unambiguously identified.

5.5.3 Course of the 2006–2009 Bluetongue Epidemic in Germany

Until the end of 2006, a total of 890 cases/outbreaks were reported from the German federal states of North Rhine-Westphalia, Hesse, Rhineland-Palatinate and Lower Saxony to the German Animal Disease Notification System (TierseuchenNachrichtenSystem, TSN; accessed 23/11/2011). Between January and April 2007, when no transmission was expected, 185 further outbreaks were reported (Table 5.1). It is likely that at least the vast majority of infected animals detected in winter 2006/2007 had contracted BT in summer or autumn 2006.

Table 5.1 Number of reported outbreaks 2006/2007 and affected species
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By analysing the outbreak data as reported to the TSN database and comparing them to the number of animals kept on the affected farms, it became apparent that at least 67,080 cattle, 9,825 sheep and 56 goats were present on premises affected by BTV-8 between August 2006 and April 2007 (Table 5.3). Of these animals, 1,529 cattle (2.28%) and 592 sheep (6.03%) were found infected. Eighty-four cattle and 222 sheep died. The case-fatality rate was much higher in sheep (37.5%) than in cattle (5.5%). These calculations are based on the assumption that all BT cases were reported. Since the infections caused only mild disease or remained even clinically inapparent in some animals, in particular cattle, it is likely that there was a substantial level of underreporting. As a consequence, the case-fatality rate in cattle might be slightly overestimated (Conraths et al. 2009).

Apparently, BTV-8 overwintered in the region and flared up again in 2007 to spread over most of western Germany during summer and autumn 2007. The first outbreak after the end of the cold season was confirmed in June 2007 in the district Oberbergischer Kreis, North Rhine-Westphalia, when a sentinel animal (cattle) sampled in May 2007 tested positive (Hoffmann et al. 2008).

The infection also re-emerged in other European countries that had been affected in 2006. BT spread rapidly through Belgium, the Netherlands, Germany, France and Luxembourg and reached the Czech Republic, Denmark, Italy, Spain, Switzerland and the United Kingdom.

Between May 2007 and April 2008, more than 22,600 cases/outbreaks were reported from Germany (Table 5.2; Conraths et al. 2009; Gethmann et al. 2010). Due to the enlargement of the affected territory in 2007, the exposed population of animals kept in farms with BT cases rose to at least 1,501,994 cattle, 505,934 sheep and 3,736 goats. The number of diseased animals on these farms amounted to 33,839 cattle, 32,158 sheep and 227 goats (Table 5.3). While mortality remained at relatively low levels as in 2006, the case-fatality rate rose to 10.8% in cattle and 41.5% in sheep.

Table 5.2 Number of reported outbreaks 2007/2008 and affected species
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Table 5.3 Exposed, diseased and dead animals and morbidity, mortality and case-fatality rate of BT infections for Germany in 2006 and 2007
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To take potential underreporting of BT cases into account, we also determined the number of animals for which the owners received financial aid from the German animal disease compensation funds (Tierseuchenkassen). In total, 10,240 cattle, 33,233 sheep and 102 goats were compensated for in 2007. This indicates an overall mortality of 0.08% in cattle and 1.36% in sheep. By focussing on the core region (North Rhine-Westphalia), where a prevalence of up to 100% can be assumed (by comparison to the situation in Belgium; Gethmann et al. unpublished), the mortality was 0.51% in cattle and 13.19% in sheep.

While several member states declared vector-free periods according to Commission Regulation (EC) No. 1266/2007 of 26 October 2007 during the cold season, i.e. defined as a period where the risk of virus transmission is deemed extremely low or negligible, thus allowing for a temporary lift of some trade restrictions, transmission was shown in Schleswig-Holstein in February 2008 (Hoffmann et al. 2008). This indicates that vector transmission on a low level may have played a role in the overwintering mechanism of BT in Germany and supported the view that declaration of a seasonally vector-free period was not appropriate for Germany.

In order to control BTV-8, to reduce the suffering of BT-infected animals and to mitigate the economic damage caused by the epizootic, it was decided to conduct a compulsory vaccination programme in Germany using inactivated vaccines which had not yet been licensed when the programme started (for details, see Chap. 5.6.2 Vaccination). The vaccination programme started in May and led to a massive decrease of new outbreaks in 2008 (Conraths et al. 2009) (Fig. 5.7, Table 5.4).

Fig. 5.7
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Bluetongue disease, monthly incidence (source: TSN)

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Table 5.4 Number of reported outbreaks 2008/2009
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Between May and December 2008, a total of 3,083 new BTV-8 outbreaks plus 19 BTV-6 cases were reported (Table 5.4). They were mainly found in two regions in the north-west of Lower Saxony and Western parts of Baden-Württemberg. These cases can be explained by the relatively late onset of the immunization campaign because of initially limited supply of BTV-8 vaccines.

At the end of 2008, the genome of BTV serotype 6 (BTV-6) was detected in the district of Grafschaft Bentheim in BTV-1-vaccinated animals. In November and December, outbreaks in a total of 19 cattle farms were reported (Eschbaumer et al. 2010a). None of the animals showed clinical symptoms. Similar cases had previously been reported in the Netherlands (presentation at SCFCAH, section animal health and animal welfare, 08 December 2008, http://ec.europa.eu/food/committees/regulatory/scfcah/animal_health/presentations_en.htm). Despite comprehensive epidemiological investigations, the source of infection could not be identified. It cannot be excluded that animals were illegally vaccinated with an imported modified-live vaccine and that spread vaccine virus may have reassorted (Saegerman and Pastoret 2009). In 2009, no further cases of BTV-6 were detected despite intensive monitoring.

Since May 2009, only 12 outbreaks of BTV-8 in 9 cattle herds and 3 sheep flocks were reported, the last one occurred in November 2009 (TSN; accessed 23/11/2011).

5.6 Control Measures

After the first occurrence of BTV-8, Germany carried out the measures according to “Council Directive 2000/75/EC of 20 November 2000 laying down specific provisions for the control and eradication of bluetongue” in combination with national legislation (Verordnung zum Schutz gegen die Blauzungenkrankheit, Verordnung zum Schutz vor der Verschleppung der Blauzungenkrankheit). The measures focussed on (1) outbreak investigations in combination with monitoring and surveillance, (2) establishing restriction zones (e.g. movement restrictions) and (3) treating affected animals, farms, etc., with insecticides. In 2006, zones with a radius of at least 20 and 150 km were established around each outbreak farm (Fig. 5.8). In October 2007, measures regarding control, monitoring, surveillance and restrictions on movements of certain animals of susceptible species in relation to bluetongue were specified in Commission Regulation (EC) No 1266/2007 of 26 October 2007.

Fig. 5.8
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Outbreaks and restriction zones in Germany by the end of April 2007

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5.6.1 Movement Restrictions

Historically, BT has been regarded as an “exotic” disease in Europe, although sporadic incursions were observed on Cyprus in the first half of the twentieth century and in the mainland of Europe since the 1950s (Wilson and Mellor 2009). As a consequence, a protection zone with a radius of at least 100 km around the infected holding and a surveillance zone with a depth of at least 50 km extending beyond the limits of the protection zone were provided for (Council Directive 2000/75/EC of 20 November 2000 laying down specific provisions for the control and eradication of bluetongue). An exit ban on animals and an epidemiosurveillance programme based on the monitoring of sentinel groups preferentially of bovine animals and of vector populations were imposed for both zones. While vaccination against BT can be allowed in the protection zone, animals must not be immunized against the disease in the surveillance zone. Trade within the same zone was allowed, but there were strict limitations for moving animals from one zone to another according to defined criteria (Commission Decision 2005/393/EC, replaced by Commission Regulation (EC) No 1266/2007), such as testing blood samples of the animals for BTV before movement, protecting them against vectors and treating them against insects prior to movement (Hateley 2009).

Since all control measures, including restrictions of animal transport, use of insecticides and indoor keeping of animals, had only limited effect during the BTV-8 epidemic that started in Belgium, Germany and the Netherlands in August 2006 (Mintiens et al. 2008b), vaccination of susceptible species with inactivated vaccines was included as the method of choice for BT control as soon as BTV-8-specific vaccines became available.

5.6.2 Vaccination

In general, vaccination is the only reliable means to protect animals from clinical bluetongue disease, while at the same time preventing the onward transmission of the virus. The two most common types of BTV vaccines are attenuated modified-live viruses and inactivated whole virus preparations with adjuvants. For modified-live vaccines, there is a delicate balance between achieving an acceptable reduction in virulence while at the same time maintaining the required level of immunogenicity (Alpar et al. 2009). In adverse circumstances, live vaccines can cause disease (Veronesi et al. 2005, 2010; Monaco et al. 2006), can be transmitted by vectors (Ferrari et al. 2005; Listes et al. 2009) and can exchange genetic information with field strains (Batten et al. 2008b; Maan et al. 2010). The repeated culture passages used for attenuation alter the tissue tropism of the virus; this can lead to teratogenic effects in pregnant animals (Kirkland and Hawkes 2004; Maclachlan et al. 2009). Hence, when BTV-8 was introduced to Europe in 2006, concerns over the safety of live vaccines prevented their use. The affected countries opted to wait until highly effective and safe inactivated vaccines became available in 2008 (Eschbaumer et al. 2009; Gethmann et al. 2009). A large-scale vaccination campaign all across Europe eventually brought the epizootic to a hold and it appears as if BTV-8 has been eradicated (Zientara et al. 2010). Since the end of mandatory vaccination in 2010, however, coverage is decreasing; even though the inactivated vaccines afford good long-term protection (Wäckerlin et al. 2010; Oura et al. 2012), replacement of stock will eventually return the animal population to its initial vulnerable state (Gethmann et al. 2010).

Infection with one serotype does not lead to cross-protective immunity, and neither does vaccination with a monovalent vaccine (Alpar et al. 2009; Bréard et al. 2011; Eschbaumer et al. 2011b). It has been shown experimentally, however, that sequential infection with several serotypes can give rise to neutralizing antibody against others (Jeggo et al. 1983). This suggests that broadly protective vaccines are possible, but none have been developed so far.

Another issue with currently available vaccines is the absence of a reliable strategy to differentiate infected from vaccinated animals (DIVA) (van Oirschot 1999; Mertens et al. 2009). Experimental vector vaccines (based on pox and herpes viruses) have shown some potential (Wade-Evans et al. 1996; Lobato et al. 1997; Boone et al. 2007; Calvo-Pinilla et al. 2009a; Franceschi et al. 2011). Since these vaccines only elicit an immune response to a subset of BTV proteins, the absent proteins can serve as negative markers in a DIVA strategy. The same goes for virus-like particles (Roy et al. 1994; Stewart et al. 2010), in vitro-assembled virus capsids without genome that do not evoke antibodies to non-structural proteins. Building on the reverse genetics system for BTV (Boyce et al. 2008), upcoming disabled infectious single-cycle vaccines (Matsuo et al. 2011) could combine the excellent immunogenicity of modified-live vaccines, the safety of inactivated vaccines and the DIVA capability of vector vaccines, if suitable companion tests are developed.

After the first introduction of BTV-8 to Belgium, Germany and the Netherlands in 2006, the massive spread of BTV-8 in 2007, reports about a large number of diseased and dead animals, and the failure of other control measures, the commission and the member states decided to carry out an harmonized vaccination programme to control BT (EU 2008: Bluetongue: Commission offers co-funding for vaccination campaign, Press release, IP/08/51, Brussels, 16 January 2008). Since the available vaccines against BTV-8 had not been registered at this time and safety and efficacy assessments were rudimentary, a large-scale safety study in combination with an efficacy study was conducted before a compulsory vaccination campaign involving the administration of millions of doses of largely untested BTV-8 vaccine was started in Germany. Participation in the study was prerequisite for a temporary emergency authorization to be granted by German law (Tierseuchengesetz §17c). Three monovalent inactivated BTV8 vaccines, precisely BLUEVAC® 8 (CZ Veterinaria), BTVPUR® AlSap 8 (Merial), and Zulvac® 8 Ovis or Bovis, respectively (Fort Dodge), were tested and proved to be safe and efficacious (Gethmann et al. 2009; Eschbaumer et al. 2009; Wäckerlin et al. 2010). For the basic immunization, administration of two doses was necessary in cattle, while a single dose was deemed sufficient in sheep and goats.

The first batches of the vaccines were delivered in May 2008. Until the end of 2008, about 20 million doses were administered to cattle and 2.6 million doses to sheep. In 2009, 13 million doses were applied to cattle and 2.1 million doses to sheep, so that the vaccination coverage was over 80%. In 2009, a further vaccine, Bovilis® BTV8 (Intervet), was introduced in the vaccination programme. By the end of 2009, the German federal states decided by majority vote to switch from a compulsory vaccination programme to a voluntary programme, resulting in a decrease of the administered vaccine doses. Only 5 million doses in cattle and 0.6 million doses in sheep were reported to the national animal database (HI Tier).

It has been pointed out, however, that low vaccination coverage or the introduction of other serotypes could result in further, potentially severe outbreaks in the future (Szmaragd et al. 2010).

5.6.2.1 Claims of Potential Adverse Reactions

Although the application of BTV-8 vaccines might induce moderate, short-term local inflammatory reactions at the injection site and a transient rise in body temperature shortly after booster vaccination, the vaccines proved to be well tolerated by both cattle and sheep (Bruckner et al. 2009; Gethmann et al. 2009). However, despite the proof of the safety of the vaccines, farmers, especially in south-eastern Germany and Switzerland, claimed a wide range of adverse reactions during the compulsory vaccination programme in 2008/2009, including reduction in milk yield, increase of somatic cell count in milk, mastitis or alterations of milk quality, reduced fertility and abortions. Officially, a total of 616 adverse reactions were reported in Germany to the Federal Agency for Vaccines and Biomedicines, thereof 547 in cattle (Hoffmann and Cußler 2009). In Switzerland, a total of 1,000 reports related to BTV-8 vaccination were received in 2009, the most frequently reported suspected adverse reactions being abortion, mastitis or alterations of milk quality (Müntener et al. 2010). However, in both countries evaluation of the data showed that plausible links between vaccination and the suspected adverse reactions could not be demonstrated (Probst et al. 2011; Tschuor et al. 2010). In any case, compared to the negative effect of BTV exposure on fertility, the possible side effect of vaccination seems to be rather small and therefore should not be an obstacle to vaccination (Nusinovicia et al. 2011).

5.6.3 Vector Evasion and Control

Vector evasion strategies and the use of repellents and insecticides alone are unlikely to lead to effective BT control (Mullens et al. 2001; EFSA 2007), although they may reduce vectorial capacity, i.e. reducing attack rates and the survival of adult midges (Mullens 1992). Vector evasion and control measures may thus be useful as auxiliary or mitigation measures which should be preferably applied in addition to vaccination against the relevant serotypes of BTV, the method of choice for the control of BT.

5.6.3.1 Vector Evasion Measures

It has been suggested that simple husbandry changes and practical midge control measures may help to diminish the risk of infection for susceptible animals, e.g. by housing livestock during times of maximum midge activity (from dusk to dawn) to reduce biting rates and thus transmission of BTV

Culicoides midges that carry BTV are believed to breed on animal dung and moist soil, either bare or covered in short grass. Identifying breeding grounds and breaking the breeding cycle may thus reduce the local midge population and hamper virus transmission.

It has been proposed that turning off taps, mending leaks and filling in or draining damp areas might also help to dry up breeding sites and that dung heaps or slurry pits should be covered or removed, and their perimeters regularly scraped to remove or destroy developing larvae of biting midges. Although it seems plausible that these measures might have an effect, published studies demonstrating their efficacy are lacking.

5.6.3.2 Use of Insecticides for Vector Control

Pyrethrum and synthetic pyrethroids are the most important compounds used against biting midges. They combine a repellent activity with toxic effects on insects. Due to the high efficiency of transmission of BTV from the biting midge to the vertebrate host, the repellent effect is particularly relevant for preventing BT infections as it may protect hosts from vector bites. However, an extremely high efficacy of the repellent seems to be required to achieve a significant level of protection against BT infections. Since the repellent activity of pyrethroids decreases much quicker than their toxic effect, it is difficult to take advantage of the repellent activity without applying the compounds repeatedly in short intervals. Frequent application may however increase the risk of side effects and lead to an unacceptable impact on non-target insects such as bees, beetles, etc.

Other potentially suitable compound groups include macrocyclic lactones, organophosphates, carbamates, chloronicotinyls (e.g. imidacloprid) and phenylpyrazoles (fipronil).

The efficacy of various pyrethroids against Culicoides spp. has been examined in several studies. Depending on the specific product and its formulation, deltamethrin, permethrin, cyfluthrin and cypermethrin protected animals for 3–5 weeks (Mehlhorn et al. 2008a, b; Liebisch and Liebisch 2008; Liebisch et al. 2008a, b; Papadopoulos et al. 2009; Schmahl et al. 2008, 2009a–c; Mullens et al. 2010). It is important to note that the treated animals were only protected if a sufficient concentration of the compound near the predilection sites of biting midges was warranted.

5.6.3.2.1 Pour-On and Spot-On Formulations of Repellents and Insecticides

Pour-on and spot-on formulations of repellents and insecticides have been successfully used against biting midges. It should be noted that pour-on and spot-on treatments with permethrin and deltamethrin lead to a dorsoventral gradient of the compound concentration, also after correct application of the product, with the consequence of a reduced insecticidal effectiveness in the bioassay (Mullens et al. 2000, 2001; Liebisch et al. 2008a). Moreover, a field study conducted in Brandenburg, Germany, showed that a regular pour-on treatment of bulls in intervals of 6 weeks had neither an effect on the total number of biting midges caught in a UV trap nor on the number of blood-fed midges (Bauer et al. 2009).

5.6.3.2.2 Nets

Fine mesh nettings and fabrics impregnated with insecticide, in particular pyrethroids, have been proposed to protect stables or to reduce contact of livestock with potentially infected midges (Braverman 1989; Carpenter et al. 2008) and were recently evaluated under field conditions (Bauer et al. 2009; Skrock et al. 2010; Skrock 2011). When Meiswinkel et al. (2000) gauzed all windows of a stable with a fine mesh screening and kept the doors closed, a 14-fold reduction in the number of Culicoides entering the stable was achieved. This approach may be useful as it reduces the biting rate. It is also a cheap measure that is easy to implement and requires only little maintenance.

5.6.3.2.3 Ear Tags

Holbrook (1986) treated cattle with one fenvalerate ear tag per animal and exposed adult C. variipennis in the laboratory to hair clippings recovered from the animals on several days post treatment. The insecticidal activity on the biting midges lasted throughout the 70-day test period, with decreased efficacy following rainfall and after the 49th test day. The duration of the repellent and toxic effect of ear tags may be shorter than that of pour-on or spot-on formulations and depend on the number of ear tags used per animal. For permethrin, a toxic and repellent activity of up to 7 days was observed if a single ear tag was applied, while the effects of two ear tags lasted for up to 19 days (Liebisch et al. 2008b). The repellent effect of cypermethrin on C. sonorensis lasted for 3–5 weeks (Reeves et al. 2010), while Liebisch and Liebisch (2008) determined an insecticidal effect of 14 (one ear tag) to 21 days (two ear tags).

5.6.3.2.4 Dipping

While this method has been successfully applied in many regions of the southern hemisphere including African and South American countries as well as Australia, farms in central and northern Europe are rarely equipped with dips, and the required formulations of pyrethroids or organophosphates are hardly available or their use has been suspended due to their environmental impact (pyrethroids) or discouraged because of their toxicity to users (EFSA 2007).

5.6.3.2.5 Systemic Use of Insecticides

The use of injectable macrocyclic lactones against biting midges yielded variable results (Standfast et al. 1985; Holbrook 1994; Holbrook and Mullens 1994). Since these compounds have no repellent, but only a toxic effect on insects, they may only act by reducing the population density of biting midges due to their effect on adult stages and on the larvae of dung-inhabiting Culicoides, thus reducing the vectorial capacity in a limited area surrounding the treated animals (EFSA 2007). A direct effect on BT transmission by reducing the biting rate of the relevant vectors cannot be expected.

5.7 Economic Impact

The financial impact of BTV-8 in the Netherlands including production losses, diagnosis, treatment and disease control amounted to 32 million Euros in 2006, 164–175 million Euros in 2007 (Velthuis et al. 2010) and about 41 million Euros in 2008 (Velthuis 2011).

The Animal Health Service of North Rhine-Westphalia, Germany, calculated mean production losses of 197 € per cow for a farm with 25 cows (http://www.landwirtschaftskammer.de/landwirtschaft/tiergesundheit/rgd/index.htm).

These data were used as reference for estimating the financial impact of BTV-8 for Germany as a whole. Outbreak data from the German animal disease notification system, information on payments made by the German animal disease compensation funds (Tierseuchenkassen) to BT-affected farms and administrative data were also included in the analysis. According to these calculations, the financial losses BTV-8 caused in Germany amounted to approximately 31 million Euros in 2006, 90 million Euros in 2007, 89 million Euros in 2008, 35 million Euros in 2009 and 11 million Euros in 2010, i.e. a total loss of 254 million Euros so far (Table 5.5). It has to be taken into account, however, that some parameters (e.g. morbidity, trade costs) could not be calculated exactly but had to be estimated.

Table 5.5 Economic losses in euros caused by the bluetongue disease epidemic in 2006–2009
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